The actin and microtubule cytoskeletons regulate critical aspects of T cell biology including development, migration and activation through the organization and stabilization of signaling complexes at the immunological synapse (IS). Likewise, proteins that regulate the cytoskeleton play an equally fundamental role in immune regulation. This renewal application focuses on the newly identified Wiskott - Aldrich syndrome protein (WASP) family member, WASH (WASP and SCAR homologue). Our recent Developmental Cell paper shows that WASH localizes to endosomal subdomains in cells, interacts with microtubules and regulates sorting events at endosomes, in particular transport of cargo from endosomes to the Golgi by the retromer (a complex of proteins involved in sorting receptors from the early/late endosome to the trans-Golgi network), in an Arp2/3- and microtubule-dependent manner. Actin regulatory functions of WASP and WAVE proteins are controlled by unique N-terminal regions, which mediate their incorporation into distinct protein complexes linked to diverse biological functions. Thus, understanding the biochemical make-up and mechanisms that regulate assembly, localization and activity of these cytoskeletal regulators is paramount to understanding their cellular functions. Our preliminary data also demonstrate that WASH exists in a multi-protein complex containing 4 previously uncharacterized proteins (FAM21, SWIP, Strumpellin and CCDC53). Accordingly, a central focus of this proposal is the mechanisms of WASH complex assembly and the mechanisms that control its localization, activity and microtubule interaction (Aim 1). An emerging paradigm suggests that the dynamic endosomal tubulovesicular network is not just involved in receptor degradation and recycling, but is also a site at which receptor signaling can be initiated, sustained or terminated. Our published data indicate that WASH localizes to endosomal subdomains where it regulates endosomal sorting. We now provide preliminary data showing that the WASH complex interacts with several signaling proteins following TCR ligation, localizes on endosomal subdomains following TCR ligation with PLC31, and depletion of the WASH complex attenuates TCR- stimulated signaling including PLC31 activation in DP thymocytes. Taken together, our data suggest that WASH regulates endosomal-sorting pathways involved in TCR-mediated activation and selection. These mechanisms will be examined in Aim 2. In two highly focused aims we will address the hypothesis that WASH functions as part of a macromolecular complex to coordinate actin and microtubule-dependent vesicular trafficking/sorting, which quantitatively regulates TCR signaling leading to distinct T cell fates and functions. The strength of the TCR signal is a critical checkpoint in the regulation of thymocyte selection, T cell activation and effector function. Therefore understanding the regulation of TCR signaling by WASH will provide insight into novel mechanisms of immune regulation. Our proposed studies are outlined in the two Specific Aims below. Results from these studies will enhance our understanding of the mechanisms by which TCR signaling is regulated and will provide insight into novel pathways that could potentially be manipulated to ameliorate immunodeficiency diseases and/or autoimmune/inflammatory conditions. PUBLIC HEALTH RELEVANCE: The actin and microtubule cytoskeletons participate in multiple aspects of T cell biology including development, migration, effector functions and the organization and stabilization of signaling complexes at the immunological synapse. Consequently, proteins that regulate cytoskeletal-dependent processes play a fundamental role in immune regulation. The experiments in this proposal are aimed at understanding the role of the novel actin regulatory protein WASH in T cell activation and thymocyte selection.